CN102243233A - Immunological detection method and kit for detecting mycobacterium tuberculosis - Google Patents
Immunological detection method and kit for detecting mycobacterium tuberculosis Download PDFInfo
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- CN102243233A CN102243233A CN2010101718675A CN201010171867A CN102243233A CN 102243233 A CN102243233 A CN 102243233A CN 2010101718675 A CN2010101718675 A CN 2010101718675A CN 201010171867 A CN201010171867 A CN 201010171867A CN 102243233 A CN102243233 A CN 102243233A
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Abstract
The invention discloses an immunological detection method and a kit for detecting mycobacterium tuberculosis. According to the immunological detection method, mycobacterium tuberculosis specific antigens are adopted. The mycobacterium tuberculosis specific antigens comprise one or two components selected from protein 38kDa, protein CFP10, carbohydrate antigen LAM, and carbohydrate antigen TBGL. According to the invention, a multiple antigen composition is adopted as a mixed antigen for detecting tuberculosis antibodies. The method and the kit have advantages of high sensitivity, high specificity, and low false positive rate. During a real operation process, a multiple point combined determination result is improved into a single point determination result. Therefore the operation is more convenient. With the method and the kit, antibodies in tuberculosis patient body fluid samples such as serum and hydrothorax can be detected specifically in half a day. The kit can be produced in large scale with low cost. Therefore, the invention has an important significance in the controlling of tuberculosis.
Description
Technical field
The invention belongs to biological medicine external diagnosis reagent field, particularly a kind of immunological detection method of Much's bacillus and kit thereof.
Background technology
Diagnose Much's bacillus (MTB) to infect fast and accurately, particularly the diagnosis of bacterium yin constipation nuclear is significant for control lungy.MTB diagnosis of infection method has a lot, and at present clinical method the most commonly used still relies on traditional phlegm smear bacteriology checking, but recall rate is low; MTB cultivates " goldstandard " that can make a definite diagnosis as tuberculosis, but its incubation time is oversize, in general 4-6 week, is difficult to meet clinical needs; Though the Bactec technology has shortened incubation time, expense is higher, is difficult in the short time popularize; X line and CT examination only provide the diagnosis of iconography possibility; Polymerase chain reaction (PCR) technology and other gene diagnosis method remain at complicated operation as clinical diagnosis, and be higher to technology and personnel specialty competency profiling, and still can not be used for the cloudy quick diagnosis lungy of bacterium.And the sero-immunity that MTB infects diagnosis with its intrinsic easy and simple to handle, quick, with the naked eye judged result, good stability, be convenient to popularization, need not advantage such as special exact instrument, be that most probable satisfies the diagnosis of tuberculosis technology that economically less developed region and tuberculosis district occurred frequently are needed badly under the new situation.Be based upon the simple combination on empirical basis and existing tuberculosis immunodiagnosis product is many with single antigen or minority, all can not solve the problem that susceptibility is lower, specificity is relatively poor.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is exactly at the sensitivity of existing Much's bacillus immunologic detection method existence and the deficiency of poor specificity, a kind of immunological detection method and kit thereof of Much's bacillus are provided, this method sensitivity and specificity height have wide application prospect.
The present invention solves the problems of the technologies described above one of technical scheme of being adopted: a kind of immunological detection method of Much's bacillus, comprise and use the Much's bacillus specific antigen, wherein said Much's bacillus specific antigen comprises: albumen 38kDa, PROTEIN C FP10, and among carbohydrate antigen LAM and the carbohydrate antigen TBGL one or both.
Among the present invention, described albumen 38kDa, the accession number of its sequence in public database NCBI GeneBank is (GI): 57116801 (that is: Rv0934, GeneID:885724).Described PROTEIN C FP10, the accession number of its sequence in public database NCBI GeneBank is (GI): 15611010 (be Rv3874, GeneID:886194.) described albumen 38kDa, CFP10 can be the albumen that extracts from natural Much's bacillus, the recombinant protein that perhaps utilizes gene engineering method to obtain.
Among the present invention, described carbohydrate antigen LAM can be conventional carbohydrate antigen LAM, and preferable is got by the method preparation that may further comprise the steps:
With the broken bacterium of Much's bacillus water or buffer solution suspension back of deactivation, centrifuging and taking supernatant;
With isopyknic phenol/chloroform (1: 1, the supernatant water is got in v/v) extracting;
With the chloroform/methanol of 4 times of volumes (2: 1, the supernatant water is got in v/v) extracting;
Add DNase I and RNase, remove nucleic acid;
Add Triton-X 114, put 4 ℃ and make it form protomere, 37 ℃ of water-bath layerings;
Extracting goes out the water that the upper strata does not contain detergent, adds Triton-X114, and 4 ℃ are spent the night and make it form protomere, extracting organic phase;
Lower floor contains in the organic phase of detergent and adds PBS, and 4 ℃ of processing make it form protomere, extracting organic phase after 37 ℃ of water-baths;
Organic phase is merged the back dialysis, and freeze-drying promptly gets carbohydrate antigen LAM.
The molecular weight of carbohydrate antigen LAM is at the 37.5kDa place.
Among the present invention, what described carbohydrate antigen TBGL was preferable is got by the method preparation that may further comprise the steps:
The broken bacterium of Much's bacillus water or buffer solution suspension back with deactivation;
The adding chloroform/methanol (2: 1, v/v) extracting goes out organic phase, dries up;
Adding chloroform/methanol/water (4: 2: 1, v/v/v) organic phase is got in washing;
After drying up, organic phase is dissolved in chloroform;
Cross 60～100 purpose silicagel columns, use respectively chloroform, chloroform/methanol (19: 1, v/v) and chloroform/methanol (9: 1, v/v) wash-out is collected the chloroform/methanol part, freeze-drying promptly gets carbohydrate antigen TBGL.Dying colour developing by silver identifies.
The immunological detection method of Much's bacillus of the present invention, preferable may further comprise the steps:
1) with described Much's bacillus specific antigen bag by solid phase carrier,
2) add the sample to be checked that dilutes, insulation is hatched, and clean and remove any unconjugated composition,
3) adding is through the second antibody of anti-people's antibody of mark, and insulation is hatched, and clean and remove any unconjugated composition,
4) detect immune existence in the mixtures incubated in conjunction with compound.
Among the present invention, Much's bacillus specific antigen described in the step 1) to the bag of solid phase carrier by concentration preferable be: carbohydrate antigen LAM is 0.2-8.0 μ g/ml, carbohydrate antigen TBGL is 0.1-2.0 μ g/ml, and albumen 38kDa is 0.1-1.0 μ g/ml, and PROTEIN C FP10 is 0.5-4.0 μ g/ml.Better, described Much's bacillus specific antigen is combination 1, wherein: carbohydrate antigen LAM, 0.5 μ g/ml; Albumen 38kDa, 0.8 μ g/ml; PROTEIN C FP10,0.5 μ g/ml; Perhaps make up 2, wherein: carbohydrate antigen TBGL, 2 μ g/ml; Albumen 38kDa, 1 μ g/ml; PROTEIN C FP10,0.3 μ g/ml.Described solid phase carrier can be this area solid phase carrier commonly used, and as glass, plastics etc., preferably tygon orifice plate is as 96 orifice plates etc.
Among the present invention, step 2) sample to be checked described in can be serum or other body fluid or the tissue specimen from people to be checked, preferably from people's to be checked serum.
Among the present invention, described in the step 3) through the preferably anti-human IgG antibody of horseradish peroxidase-labeled of the second antibody of anti-people's antibody of mark, also can be the antibody of other antibody of anti-people of other mark such as IgM etc.
Among the present invention, the condition that described insulation is hatched is the condition of the suitable antigen-antibody combination of routine, preferably 15-50 ℃ of general temperature, and the time is 2 minutes-5 hours, best is 37 ℃, 0.5～2 hour.
Among the present invention, the method that any unconjugated composition is removed in described cleaning is conventional method, generally adopts the PBST washing for several times.
Among the present invention, immunity is in conjunction with preferable employing ELISA (the enzyme-linked immunosorbent assay of method of the existence of compound in the detection mixtures incubated described in the step 4); Enzyme-linked immunosorbent assay) method also can be the detection method of other routine.
The present invention solves the problems of the technologies described above two of the technical scheme that adopted: a kind of immunology detection kit of Much's bacillus, comprise the Much's bacillus specific antigen, wherein, described Much's bacillus specific antigen comprises: albumen 38kDa, PROTEIN C FP10, and among carbohydrate antigen LAM and the carbohydrate antigen TBGL one or both.
Among the present invention, described kit is preferable also comprises second antibody through anti-people's antibody of mark.Better also comprise any following one or more:
The carbonate bag of coating buffer: 0.05M is cushioned liquid (pH9.6);
Solid phase carrier: tygon orifice plate;
Cleansing solution: PBST;
Second antibody through anti-people's antibody of mark: the anti-human IgG antibody of peroxidase labelling.
Dilution: the PBST that contains 1%BSA;
Positive control serum or negative control sera;
Stop buffer: 2M sulfuric acid;
Substrate solution: substrate buffer solution A: sodium acetate 2.4g, citric acid 0.28g is dissolved in the 88ml ultrapure water, and the abundant stirring and dissolving of magnetic stirring apparatus adds 53 μ l 30%H at last
2O
2, the abundant stirring and dissolving of magnetic stirring apparatus and getting; Add 0.03g TMB and 0.32g EDTA-Na2 in the substrate buffer solution B:80ml ultrapure water, and then add 8ml glycerine, magnetic agitation makes its abundant stirring and dissolving and gets.
Among the present invention, but above-mentioned optimum condition combination in any on the basis that meets this area general knowledge promptly gets the preferred embodiments of the invention.
Raw material that the present invention is used or reagent except that specifying, all commercially available getting.
The present invention is except that specifying, used number percent all is percentage by weight.
Positive progressive effect of the present invention is:
A. to adopt the cooperation of many antigen group be the hybrid antigen of tuberculosis antibody test in the present invention.Preparation carbohydrate antigen LAM and TBGL purify from the mycobacterium tuberculosis complex bacterial strain; By technique for gene engineering clone, expression, purifying Much's bacillus recombinant protein (38kD, CFP10); New carbohydrate antigen LAM and the TBGL that extracts has Much's bacillus bacterial classification and mycobacterium Pseudomonas specificity, recombinant protein 38kDa, the CFP10 of clonal expression is RD disappearance district coding, thereby they all can guarantee higher specificity, have guaranteed the sensitivity of diagnosis.
B. the present invention is in hybrid antigen, and each single antigen has different concentration.By multiple different bags by concentration associating, reach the high sensitivity and the specificity of diagnosis.
C. the present invention takes the mode of hybrid antigen bag quilt, becomes the single-point judged result by multiple spot combination judged result in the practical operation of diagnosis, thereby convenient and do not influence susceptibility and specificity.
D. key reagents of the present invention-Much's bacillus reorganization 38kDa, CFP10 proteantigen can be mass-produced, and cost is relatively low.This kit can expressly detect antibody in the humoral samples such as tuberculosis patient serum, hydrothorax, only needs time half a day.
E. adopt the present invention to detect tuberculosis, sensitivity and specificity height, false positive rate is low.
F. adopt the present invention to detect tuberculosis, easy and simple to handle, quick, with the naked eye judged result, good stability, be convenient to promote, need not special exact instrument, significant for control lungy.
Embodiment
Further specify the present invention with embodiment below, but the present invention is not limited.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer." room temperature " described in the present invention is meant the indoor temperature of operation room, generally is 15 ℃.
Embodiment 1
1, the purification of carbohydrate antigen LAM
A, Luo Shi tuberculosis nutrient culture media are cultivated Much's bacillus H37Ra (ATCC 25177) 3～4 weeks for 37 ℃, collect surperficial culture, 80 ℃ of water-baths deactivation in 1.5 hours;
B, PBS washing, centrifuging and taking precipitation is weighed, and the wet bacterium of every gram suspends with 5ml PBS (pH7.4), ice-bath ultrasonic ripple fragmentation: 600W, 6s * 6s * 90 time.
C, 4 ℃ 10, centrifugal 30 minutes of 000g collects supernatant.
D, use Rotary Evaporators, 40 ℃ of evaporation and concentration are to 40ml.
E, (1: 1, v/v) extracting was 3 times with isopyknic phenol/chloroform.
F, (2: 1, v/v) extracting was 2 times, dialysed overnight with the chloroform/methanol of 4 times of volumes with the supernatant after phenol/chloroform extracting.
G, usefulness ultraviolet-visible spectrophotometer test sample OD
260nm, the nucleic acid content in the calculation sample is pressed 1.5U DNase I enzymolysis 0.8 μ g DNA and is added DNase I, and to add RNase be 20 μ g/ml to final concentration, 37 ℃ of water-baths in 2 hours, dialysed overnight;
H, in the residue extract, add Triton-X 114 to final concentration be 8% (v/v), put 4 ℃ and made it form protomere, 37 ℃ of water-bath layerings after 3 hours in 1 hour.The water that the upper strata is not contained detergent extracts, and adds Triton-X114 and is 8%, 4 ℃ to final concentration and spends the night and make it form protomere.The organic phase that contains detergent in lower floor adds PBS to Triton-X114 final concentration and is 8%, 4 ℃ and made it form protomere in 1 hour, 37 ℃ of water-baths 3 hours, extracting.Organic phase is merged the back dialysis, freeze-drying ,-20 ℃ of preservations are standby.Dye its molecular weight of colour developing at the 37.5kDa place by silver.
2, the purification of carbohydrate antigen TBGL
A, 7H9 medium culture H37Ra 4～6 weeks of bacterium;
B, 70 ℃ of 3h deactivation H37Ra thalline;
C, 10,000g, 4 ℃ of centrifugation thalline, 0.01M PBS (pH 7.2) suspends;
D, supersonic cell pulverize, 600w, 10s * 10s * 60 time;
The wet bacterium of E, every gram (the precipitation thalline before centrifugal back is broken) add the 30ml chloroform/methanol (2: 1, v/v) 50 ℃ of extracting 48h, the organic phase Nitrogen evaporator dries up;
F, add chloroform/methanol/water (4: 2: 1, v/v/v) washing;
G, organic phase are dissolved in chloroform after drying up with Nitrogen evaporator;
H, cross the silicagel column (self-chambering bed volume 20m1) of 60～100 order GeHealthcare, use respectively chloroform, chloroform/methanol (19: 1, v/v) and chloroform/methanol (9: 1, v/v) wash-out was collected the chloroform/methanol part, the freeze-drying preservation.Dying colour developing by silver identifies.
3, recombinant protein 38kD
A, target gene design of primers
Primer 38kDF:5 '-AGC
GGA TCCGTG AAA ATT CGT TTG CAT AC-3 ';
3 primer 8kDR:5 '-GCG
AAG CTTGCT GGA AAT CGT CGC GAT C-3 ';
Restriction enzyme site is respectively BamH, Hind III.
The pcr amplification of B, target gene
For touching plate, 38kDF and 38kDR are primer with Much's bacillus H37Rv genome, use the Taq enzyme, by the PCR 38kD protein gene that directly increases.PCR reaction conditions: 94 ℃ of pre-sex change 5min; (94 ℃, 30s; 55 ℃, 40s; 72 ℃, 90s) 35 circulations; 72 ℃ of insulation 7min; 4 ℃ of preservations.Reaction finishes the back and reclaims kit (Invitrogen) recovery with 1% agarose gel electrophoresis separation purpose fragment with DNA.
C, gene clone and sequencing
PCR product and cloning vector pET30a (+) (Invitrogen) spend the night with 4 ℃ of reactions of T4DNA ligase.Connect product CaCl
2Method transformed competence colibacillus cell E.coli DH5 α, containing ampicillin (ampicillin, amp) Luria-Bertani (LB) agar medium transforms dull and stereotyped going up through Amp (100mg/ml), blue hickie preliminary screening, choosing white colony then cultivates in a small amount, the extraction plasmid carries out enzyme and cuts evaluation, and the positive colony that screening obtains send by the order-checking of the handsome biotech company in Shanghai and identifies.Sequencing result and GeneBank go up the in full accord of report.
The structure of D, expression vector and the screening of engineering bacteria
Plasmid pMD-38kD and expression vector pET-30a (Invitrogen) after the order-checking evaluation are carried out BamH, Hind III double digestion, the 38kD gene behind the double digestion is connected for 16 ℃ with the T4DNA ligase with expression vector pET-30a spends the night then.Product CaCl
2Method transformed competence colibacillus cell E.coliBL21 (DE3) transforms on the flat board through Amp (100mg/ml) preliminary screening at the LB agar medium that contains the ampicillin, uses bacterium colony PCR screening positive clone then, i.e. BL21 (DE3)/pET-30a-38kD.
The abduction delivering of E, 38kD gene
The fresh bacterium colony of getting several BL21 (DE3)/pET-30a-38kD contains in the LB nutrient culture media of 100mg/ml Amp 37 ℃ of shaken cultivation in 5ml and spends the night.Next day bacterium liquid is inoculated in 10ml with 5: 100 (v/v) and contains in the LB nutrient culture media of 100mg/ml Amp, 37 ℃ are continued to be cultured to A
600About 0.6 o'clock, add IPTG to final concentration be 1mmol/L, continue to induce and collect thalline behind the 4h and carry out the SDS-PAGE electrophoresis, Coomassie brilliant blue R-250 dyeing.
The preparation of F, recombinant protein 38kD soluble analysis and inclusion body
Thalline after inducing is resuspended with 50mmol/L PBS (pH7.4), uses ultrasonic disruption 5min then, and the centrifugal 10min of 12000r/min gets supernatant and inclusion body sediment respectively.Get above-mentioned thalline, supernatant and inclusion body sediment, carry out 15%SDS-PAGE respectively, Coomassie brilliant blue R-250 dyeing is to analyze the existence of expression product.The albumen of results expression mainly exists with the inclusion body form.The thalline of collecting abduction delivering is in broken damping fluid (the 50mmol/L Tris-HCl pH8.0 of cell supersonic wave; 1mmol/LEDTA), ultrasonication, centrifugal collection inclusion body precipitation.With inclusion body lavation buffer solution (50mmol/LTris-HCl pH810; 2mmol/L urea; 015%Triton X-100) washing is three times, and the precipitation of acquisition is the inclusion body of purifying.The sample that takes a morsel carries out SDS-PAGE, Coomassie brilliant blue R-250 dyeing, and the target protein molecular weight is near 44kDa.
G, recombinant antigen purifying
Get the inclusion body of purifying, be resuspended in 20mL buffer A (6mol/L guanidine hydrochloride, 0.1mmol/LNaH
2PO
4, 0.01mol/L Tris
2Cl, pH8.0) in, stirring at room 2h, inclusion body is dissolved fully, behind the centrifugal 30min of 1000r/min, supernatant is joined the nickel affinity column of crossing with the buffer A balance (GEHealthcare), use buffer B (8mol/L urea, the 0.1mmol/LNaH of 5 times of bed volumes then respectively
2PO
40.01mol/L Tris-Cl, pH8.0), damping fluid C (the same buffer B of 10 times of bed volumes, pH6.3), 2 times of bed volume damping fluid D (same buffer B, pH5.9), 2 times of bed volume damping fluid E (same buffer B, pH4.5) wash post, use 10mL damping fluid F (containing the 250mmol/L imidazoles among the damping fluid C) that destination protein is carried out wash-out at last, substep is collected, the about 1mL of every pipe.Get each pipe and collect liquid 15 μ L, add equivalent 2 * sample-loading buffer, 37 ℃ of incubation 10min detect through 12%～15%SDS-PAGE, and each pipe that will contain destination protein (molecular weight is between the 30-46kDa, about 44.2kDa) merges.
The renaturation of I, expressing protein
Take progressively to reduce the method for urea concentration, remove the urea in the protein solution, make albumen natural renaturation in this process of sex change, earlier with the 4 ℃ of dialysed overnight of damping fluid that contain 6mol/L urea, then respectively with 4,3,2,1, the gradient of 0.5mol/L urea dialyses each more than the 4h, dialyse more than the 4h again with the PBS damping fluid at last.After dialysis finished, at 4 ℃ of centrifugal 10min of following 12000r/min, supernatant was soluble recombinant protein with protein solution.Send out the recombinant antigen concentration of measuring purifying with uv absorption, formula is:
Concentration (mg/ml)=1.55 * A
280-0.76 * A
260
Then use freeze dryer recombinant antigen is carried out freeze-drying, the packing of 0.5mg/ pipe is standby.Empirical tests purity reaches more than 95%.By the experiment of rabbit immunoserology, prove that it has immunocompetence.
4, recombinant protein c FP10
Primer P1:5 ' GGG
GGATCCATG GCA GAGATGAAGACC-3 ';
Primer P2:5 ' GCC
AAG CTTTCA GAA GCC CAT TTG CGA G-3 '.
Restriction enzyme site is respectively BamH, Hind III.
1. the amplification of genetic fragment
1.1DNA extraction
H37Rv solid culture a little and a physiological saline that contains 0.05% (v/v) Tween-20 of getting on the Russell medium boil 30min jointly, get supernatant after 10000g is centrifugal, extract kit (Invitrogen) with genome and extract the Much's bacillus genomic DNA.
1.2 amplification
With the H37Rv genomic DNA is template, is primer with P1 and P2, with archaeal dna polymerase amplification CFP10 gene.Reaction system is: sterilization deionized water 39 μ l, 10 * PCR damping fluid, 5 μ l, dNTP2 μ l, P1 1 μ l, P2 1 μ l, archaeal dna polymerase 1 μ l, dna profiling 1 μ l, totally 50 μ l reaction systems.Amplification condition is: 94 ℃ of 5min; 30 circulations: 94 ℃ of 45s, 62 ℃ of 45s, 72 ℃ of 45s; 72 ℃ of 10min.Reaction mixture reclaims kit (Invitrogen) with glue and reclaims PCR product purpose fragment after 1.5% agarose electrophoresis.
2. the expression and purification of construction of recombinant plasmid and albumen
2.1 construction of recombinant plasmid
1) preparation of the big fragment of plasmid pET32a
(1) extraction of plasmid pET32a
Extract pET32 plasmid (Invitrogen) with plasmid extraction kit (Invitrogen).
(2) preparation of the big fragment of carrier
With BamH, Hind III double digestion pET32a plasmid.Reaction conditions is: pET32a 30 μ l, and 10 * buffer, 4 μ l, BamH I 12 μ l (20U), Hind III 12 μ l (20U), deionized water 2 μ l form 40 μ l reaction systems, put 37 ℃ of water-bath 4h.Enzyme is cut potpourri glue behind 1% agarose gel electrophoresis and is reclaimed the big fragment of carrier.
2) coupled reaction of plasmid pET32a and PCR product
Reaction conditions is: PCR product 8 μ l (0.8 μ g), and the big fragment 2 μ l of pET32a (0.1 μ g) connect reagent 10 μ l, form 20 μ l reaction systems, and mixing connects 1h for rearmounted 16 ℃.
3) transformed competence colibacillus cell E.coli DH5 α
(1) product 10 μ l be will connect and mixing, ice-water bath 30min joined among the competent cell E.coli DH5 α;
(2) 42 ℃ of water-bath 2min, ice-water bath 2min;
(3) add 1ml LB nutrient solution, 37 ℃ of 150rpm recovery 40min;
(4) choose 200 μ l transformation mixtures and coat on the LA culture plate 37 ℃ of overnight incubation.
4) order-checking of recombinant plasmid is identified
Several single bacterium colonies (positive colony) on the picking LA screening and culturing base are inoculated in the LA nutrient solution, 37 ℃ of overnight incubation at random.Get bacterium liquid 1ml and be used for order-checking evaluation transformed bacteria pET32a-cfp10.Sequencing result and GeneBank go up the in full accord of report.
2.2. the extraction of recombinant plasmid
The transformed into escherichia coli DH5 α that contains the pET32a-cfp10 recombinant plasmid that order-checking is correct, with the 37 ℃ of overnight incubation of LA nutrient culture media that contain 25 μ g/ml ampicillins, plasmid extraction kit extracts recombinant plasmid.
3. recombinant plasmid transformed BL21 (DE3) expresses bacterium
Express bacterium with 2 μ l recombinant plasmid transformed BL21 (DE3), with LA nutrient culture media screening transformed bacteria, 37 ℃ of overnight incubation get recombinant expressed bacterium.
4. the abduction delivering of recombinant protein
(1) draw the adding of the recombinant expressed bacterium bacterium of 5ml liquid next day and be equipped with in the Erlenmeyer flask of 100ml LA nutrient culture media, 37 ℃ of 300rpm continue to cultivate 3～5h to OD
600After being 0.6～0.9, adding 1.0M IPTG respectively is 100mM, 500mM to final concentration, and continuation inducing culture 1～6h induces 5ml empty carrier bacterial strain bacterium in contrast with the same terms simultaneously;
(2) thalline of collection 1.5ml bacterium liquid, resuspended with 100 μ l, 1 * albumen sample-loading buffer, boil 6min, get 15 μ l samples and do the SDS-PAGE evaluation, take out gel after electrophoresis finishes,, on decolorization swinging table, shake decolouring gently with destainer then and spend the night with Coomassie brilliant blue G-250 dye liquor dyeing 1～2h, observe protein band, images acquired is also analyzed.
5. the purifying of recombinant protein
(1) fragmentation of expression bacterium
Get the expression bacterium 10000g centrifugation of above-mentioned 100ml nutrient solution, press the wet bacterium of 3ml/g and add ultrasonication damping fluid (2M Tris-HCl (pH8.0) 1ml, 0.5M EDTA 0.2ml, 100% beta-mercaptoethanol 0.039g, it is fixed molten to 100ml to add deionized water) resuspended, use 500W, ultrasonic broken bacterium 5min in the intensity frozen water of 5s * 5s.
(2) SDS-PAGE analyzes the expression of recombinant proteins form
Get above-mentioned bacteria breaking liquid 1.5ml, collect to go up cleer and peaceful precipitation respectively after centrifugal,, mainly express with soluble form with the expression-form that SDS-PAGE analyzes recombinant C FP10 albumen.Remaining broken liquid is collected the purifying that supernatant is used for recombinant protein through 10000g refrigerated centrifuge 20min.
(3) Ni-NTA protein purification post purifying
Get the inclusion body of purifying, be resuspended in 20mL buffer A (6mol/L guanidine hydrochloride, 0.1mmol/LNaH
2PO
4, 0.01mol/L Tris
2Cl, pH8.0) in, stirring at room 2h, inclusion body is dissolved fully, behind the centrifugal 30min of 1000r/min, supernatant is joined the nickel affinity column of crossing with the buffer A balance (GEHealthcare), use buffer B (8mol/L urea, the 0.1mmol/LNaH of 5 times of bed volumes then respectively
2PO
40.01mol/L Tris-Cl, pH8.0), damping fluid C (the same buffer B of 10 times of bed volumes, pH6.3), 2 times of bed volume damping fluid D (same buffer B, pH5.9), 2 times of bed volume damping fluid E (same buffer B, pH4.5) wash post, use 10mL damping fluid F (containing the 250mmol/L imidazoles among the damping fluid C) that destination protein is carried out wash-out at last, substep is collected, the about 1mL of every pipe.Get each pipe and collect liquid 15 μ L, add equivalent 2 * sample-loading buffer, 37 ℃ of incubation 10min detect through 12%～15%SDS-PAGE, find that the destination protein molecular weight is between the 7-17kDa, about 16.3kDa, and purity reaches more than 95%.By the experiment of rabbit immunoserology, prove that it has immunocompetence.
Embodiment 2
1, different bags are detected 1 by the associating ELISA of concentration
1) bag quilt: the carbonate bag with 0.05M is cushioned liquid (NaHCO
31.465g, Na
2CO
30.795g, add water and be settled to 1000ml, pH9.6) with single antigen diluent to working concentration, be mixed with composition 1, wherein: carbohydrate antigen LAM, 0.5 μ g/ml; Albumen 38kDa, 0.8 μ g/ml; PROTEIN C FP10,0.5 μ g/ml; Said composition 1 is placed 96 hole ELISA Plate, every hole 50 μ l, 4 ℃ are spent the night.
2) discard coating buffer, PBST washing 5 times dries.
3) every hole adds the PBST that 300 μ l contain 1%BSA, 37 ℃ of incubations 2 hours.
4) every hole adds the to be checked serum of 100 μ l with the PBST dilution that contains 1%BSA, 37 ℃ of incubations 0.5 hour.
5) discard serum, PBST washing 5 times dries.
6) every hole adds the goat anti-human igg antibody (Jackson company) of 100 μ l with the horseradish peroxidase-labeled of the PBST dilution that contains 1%BSA, 37 ℃ of incubations 0.5 hour.
7) discard liquid, PBST washing 5 times dries.
8) every hole adds the substrate solution of the fresh configuration of 100 μ l, 37 ℃ of lucifuge incubations 10 minutes.
Wherein, substrate solution is as follows: substrate buffer solution A is with sodium acetate (C
2H
3NaO
2) 2.4g and citric acid (C
6H
8O
7H
2O) 0.28g is dissolved in the 88ml ultrapure water, and the abundant stirring and dissolving of magnetic stirring apparatus adds 53 μ l 30%H at last
2O
2, the abundant stirring and dissolving of magnetic stirring apparatus; Substrate buffer solution B adds 0.03g TMB and 0.32g EDTA-Na in the 80ml ultrapure water
2(C
10H
14N
2Na
2O
82H
2O), and then add 8ml glycerine, magnetic agitation makes its abundant stirring and dissolving; Time spent mixes substrate buffer solution A, B with volume ratio at 1: 1, promptly obtains substrate solution.
9) every hole adds 50 μ l 2M sulfuric acid cessation reactions, and 450nm detects the OD value.
Above-mentioned steps 4) serum to be checked described in is respectively 85 routine tuberculosis patient serum and 87 routine healthy people's control serums.Experimental result sees Table 1.
2, different bags are detected 2 by the associating ELISA of concentration
1) bag quilt: with the carbonate bag of 0.05M be cushioned liquid (pH9.6) with single antigen diluent to working concentration, be mixed with composition 2, wherein: carbohydrate antigen TBGL, 2 μ g/ml; Albumen 38kDa, 1 μ g/ml; PROTEIN C FP10,0.3 μ g/ml; Said composition 2 is placed 96 hole ELISA Plate, every hole 50 μ l, 4 ℃ are spent the night.
2) discard coating buffer, PBST washing 5 times dries.
3) every hole adds the PBST that 300 μ l contain 1%BSA, 37 ℃ of incubations 2 hours.
4) every hole adds the to be checked serum of 100 μ l with the PBST dilution that contains 1%BSA, 37 ℃ of incubations 0.5 hour.
5) discard serum, PBST washing 5 times dries.
6) every hole adds the goat anti-human igg antibody of 100 μ l with the horseradish peroxidase-labeled of the PBST dilution that contains 1%BSA, 37 ℃ of incubations 0.5 hour.
7) discard liquid, PBST washing 5 times dries.
8) every hole adds the substrate solution of the fresh configuration of 100 μ l, 37 ℃ of lucifuge incubations 10 minutes.
9) every hole adds 50 μ l 2M sulfuric acid cessation reactions, and 450nm detects the OD value.
Above-mentioned steps 4) serum to be checked described in is to be respectively 60 routine tuberculosis patient serum and 46 routine healthy people's control serums.Experimental result sees Table 1.
The clinical assessment experimental study of table 1 various combination
Joint-detection 1:LAM+38kDa+CFP10 (certain chest hospital's tuberculosis patient tuberculosis antibody clinical assessment experimental study);
Joint-detection 2:TBGL+38kDa+CFP10 (certain medical institutions' tuberculosis antibody clinical assessment experimental study).
This shows, use antigen of mycobacterium tuberculosis combine detection tuberculosis patient of the present invention specificity up to 90% prerequisite under, sensitivity reaches more than 60% (table 1); The negative healthy philtrum false positive rate of PPD skin test is low, and false positive mainly appears in the tuberculosis infected students and BCG vaccination person of the PPD skin test positive.
Claims (10)
1. the immunological detection method of a Much's bacillus, comprise and use the Much's bacillus specific antigen, it is characterized in that described Much's bacillus specific antigen comprises: albumen 38kDa, PROTEIN C FP10, and among carbohydrate antigen LAM and the carbohydrate antigen TBGL one or both.
2. the method for claim 1 is characterized in that, the immunology detection of described Much's bacillus may further comprise the steps:
1) with described Much's bacillus specific antigen bag by solid phase carrier,
2) add the sample to be checked that dilutes, insulation is hatched, and clean and remove any unconjugated composition,
3) adding is through the second antibody of anti-people's antibody of mark, and insulation is hatched, and clean and remove any unconjugated composition,
4) detect immune existence in the mixtures incubated in conjunction with compound.
3. method as claimed in claim 2, it is characterized in that, Much's bacillus specific antigen described in the step 1) to the bag of solid phase carrier by concentration is: carbohydrate antigen LAM is 0.2-8.0 μ g/ml, carbohydrate antigen TBGL is 0.1-2.0 μ g/ml, albumen 38kDa is 0.1-1.0 μ g/ml, and PROTEIN C FP10 is 0.5-4.0 μ g/ml.
4. method as claimed in claim 2 is characterized in that step 2) described in sample to be checked be meant serum from people to be checked.
5. method as claimed in claim 2 is characterized in that, the second antibody described in the step 3) is meant the anti-human IgG antibody of horseradish peroxidase-labeled.
6. the immunology detection kit of a Much's bacillus, comprise the Much's bacillus specific antigen, it is characterized in that described Much's bacillus specific antigen comprises: albumen 38kDa, PROTEIN C FP10, and among carbohydrate antigen LAM and the carbohydrate antigen TBGL one or both.
7. kit as claimed in claim 6 is characterized in that, also comprises the second antibody through anti-people's antibody of mark.
8. kit as claimed in claim 6 also comprises any following one or more:
The carbonate bag of coating buffer: pH9.6,0.05M is cushioned liquid;
Solid phase carrier: tygon orifice plate;
Cleansing solution: PBST;
Second antibody through anti-people's antibody of mark: the anti-human IgG antibody of peroxidase labelling.
Dilution: the PBST that contains 1%BSA;
Positive control serum or negative control sera;
Stop buffer: 2M sulfuric acid;
Substrate solution: substrate buffer solution A: sodium acetate 2.4g, citric acid 0.28g is dissolved in the 88ml ultrapure water, and the abundant stirring and dissolving of magnetic stirring apparatus adds 53 μ l 30%H at last
2O
2, the abundant stirring and dissolving of magnetic stirring apparatus and getting; Add 0.03g TMB and 0.32g EDTA-Na2 in the substrate buffer solution B:80ml ultrapure water, and then add 8ml glycerine, magnetic agitation makes its abundant stirring and dissolving and gets.
9. the preparation method of a carbohydrate antigen TBGL may further comprise the steps:
The broken bacterium of Much's bacillus water or buffer solution suspension back with deactivation;
Add the extracting of 2: 1 (v/v) chloroform/methanol and go out organic phase, dry up;
Add (v/v/v) chloroform/methanol/water washing in 4: 2: 1, get organic phase;
After drying up, organic phase is dissolved in chloroform;
Cross 60～100 purpose silicagel columns, use chloroform, 19: 1 (v/v) chloroform/methanol and 9: 1 (v/v) chloroform/methanol wash-outs respectively, collect the chloroform/methanol part, freeze-drying promptly gets carbohydrate antigen TBGL.
10. preparation method as claimed in claim 9 and carbohydrate antigen TBGL.
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| CN102584962A (en) * | 2012-03-16 | 2012-07-18 | 河南中医学院 | Preparation method and application of mycobacterium tuberculosis Rv3117 recombinant protein |
| CN102621232A (en) * | 2012-03-27 | 2012-08-01 | 重庆大学 | Multi-field coupling large-sized simulation test system for coal mine dynamic disaster |
| CN102680684A (en) * | 2012-06-06 | 2012-09-19 | 李荣秀 | Detection method for specific whole blood thymus (T) cells of tuberculosis antigens |
| CN102718871A (en) * | 2012-06-01 | 2012-10-10 | 上海交通大学 | Recombinant protein of mycobacterium tuberculosis specific marker Rv 1284, preparation method and application thereof |
| CN103467582A (en) * | 2012-06-08 | 2013-12-25 | 同济大学 | High throughput screening of tubercle bacillus important antigens, and application of tubercle bacillus important antigens in tuberculosis diagnosis |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102584962A (en) * | 2012-03-16 | 2012-07-18 | 河南中医学院 | Preparation method and application of mycobacterium tuberculosis Rv3117 recombinant protein |
| CN102621232A (en) * | 2012-03-27 | 2012-08-01 | 重庆大学 | Multi-field coupling large-sized simulation test system for coal mine dynamic disaster |
| CN102621232B (en) * | 2012-03-27 | 2013-12-18 | 重庆大学 | Multi-field coupling large-sized simulation test system for coal mine dynamic disaster |
| CN102718871A (en) * | 2012-06-01 | 2012-10-10 | 上海交通大学 | Recombinant protein of mycobacterium tuberculosis specific marker Rv 1284, preparation method and application thereof |
| CN102680684A (en) * | 2012-06-06 | 2012-09-19 | 李荣秀 | Detection method for specific whole blood thymus (T) cells of tuberculosis antigens |
| CN102680684B (en) * | 2012-06-06 | 2014-12-10 | 李荣秀 | Detection method for specific whole blood thymus (T) cells of tuberculosis antigens |
| CN103467582A (en) * | 2012-06-08 | 2013-12-25 | 同济大学 | High throughput screening of tubercle bacillus important antigens, and application of tubercle bacillus important antigens in tuberculosis diagnosis |
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Application publication date: 20111116 |